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Keisuke Usui

Publications and source records attributed to Keisuke Usui.

4 recordsLinked to original sources

A self-contained polymeric 2-DE chip system for rapid and easy analysis.

We developed a polymeric 2-DE chip system. The chip consisted of an IEF region, an SDS-PAGE region, a valveless connection port, and a sample introduction port. A "junction structure" as a valveless connection port, which allowed separating and connecting the first- and second-dimensional gels, was fabricated between their regions. A "solution inlet" as a sample introduction port was fabricated to perform the liquid and sample introductions without solution leakage. Simultaneous sample monitoring was performed using the on-chip detection system. The performances of the system were demonstrated using commercially available proteins as a standard specimen and tissue-extracted proteins as the real samples. All procedures were employed without any movement of relocation part. This new 2-D separation system realized improved labor-intensive operations and a reduced experimental time.

Animals↗

Role of the N-terminal region of the crenarchaeal sHsp, StHsp14.0, in thermal-induced disassembly of the complex and molecular chaperone activity.

Small heat shock protein is a ubiquitous molecular chaperone, which consists of a non-conserved N-terminal region followed by a conserved alpha-crystallin domain. To understand the role of the N-terminal region, we constructed N-terminal truncation mutants of StHsp14.0, the sHsp from Sulfolobus tokodaii strain 7. All the mutants formed a stable oligomeric complex similar to that of the wild type. Electron microscopy and size exclusion chromatography-multiangle light scattering showed that the N-terminal region should locate in the center of the oligomeric particle. The mutants exhibited reduced chaperone activity for the protection of 3-isopropylmalate dehydrogenase from thermal aggregation. This reduction correlates with lowered subunit exchange efficiency. The oligomeric structure was retained even after incubation at 90 degrees C. These results suggest that the N-terminal region of StHsp14.0 functions in the thermally induced disassembly of the complex.

3-Isopropylmalate Dehydrogenase↗

Expression and biochemical characterization of two small heat shock proteins from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7.

We expressed and characterized two sHsps, StHsp19.7 and StHsp14.0, from a thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7. StHsp19.7 forms a filamentous structure consisting of spherical particles and lacks molecular chaperone activity. Fractionation of Sulfolobus extracts by size exclusion chromatography with immunoblotting indicates that StHsp19.7 exists as a filamentous structure in vivo. On the other hand, StHsp14.0 exists as a spherical oligomer like other sHsps. It showed molecular chaperone activity to protect thermophilic 3-isopropylmalate dehydrogenase (IPMDH) from thermal aggregation at 87 degrees C. StHsp14.0 formed variable-sized complexes with denatured IPMDH at 90 degrees C. Using StHsp14.0 labeled with fluorescence or biotin probe and magnetic separation, subunit exchanges between complexes were demonstrated. This is the first report on the filament formation of sHsp and also the high molecular chaperone activity of thermophilic archaeal sHsps.

3-Isopropylmalate Dehydrogenase↗